Fattori naturali e antropici che influenzano il ciclo biologico di specie di insetti esotici e nativi

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DOTTORATO DI RICERCA IN

Scienze e Tecnologie Agrarie Ambientali e Alimentari

Ciclo XXVIII

Settore Concorsuale di afferenza: 07/D1 – PATOLOGIA VEGETALE ED ENTOMOLOGIA Settore Scientifico disciplinare: AGR/11 – ENTOMOLOGIA GENERALE E APPLICATA

TITOLO TESI

Natural and anthropogenic factors affecting the

life cycle of exotic and native insect species

Presentata da: Cinzia Di Vitantonio

Coordinatore Dottorato

Relatore

Prof. Giovanni Dinelli

Prof. Maria Luisa Dindo

2

INDEX

pag

GENERAL INTRODUCTION……….

9

Chapter 1……….

12

1.LONGEVITY AND REPRODUCTION CAPACITY

OF TWO COCCINELLID SPECIES PARASITIZED

BY DINOCAMPUS COCCINELLAE (SCHRANK)

(HYMENOPTERA BRACONIDAE)

INTRODUCTION………

12

1.1 EXOTIC INSECTS AND INDIGENOUS PARASITOIDS……..

14

1.2 Harmonia axyridis (Pallas)………

16

1.3 Adalia bipunctata (L.)………

19

1.4 Myzus persicae (Sulzer)………..

20

1.5 Dinocampus coccinellae (Schrank)……….

21

2. MATERIALS AND METHODS………..

24

2.1 INSECTS USED FOR THE EXPERIMENT……….

24

3

2.1.2 D. coccinellae rearing………25

2.2 . EXPERIMENTAL TRIALS………..26

2.2.1. Parasitoid performance test………..26

2.2.2. Parameters considered in the study...27

2.3 DATA ANALYSIS………...27

3. RESULTS AND DISCUSSION……….

28

4. CONCLUSIONS……….

39

Chapter 2………

42

1. PARASITIZATION EFFECT OF THE TACHINID

EXORISTA LARVARUM (L.) ON THE EXOTIC BOX

TREE

MOTH

CYDALIMA

PERSPECTALIS

(WALKER) (LEPIDOPTERA CRAMBIDAE)

INTRODUCTION……….42

1.1 Cydalima perspectalis (Walker)………45

1.2 Exorista larvarum (L.)………

46

2. MATERIALS AND METHODS………

49

4

2.1.1 C. prespectalis rearing……….

49

2.1.2 E. larvarum rearing………...

50

2.2 EXPERIMENTAL TRIALS………

50

2.2.1. Description……….

50

2.2.2. Parameters considered in the study………

51

2.3 DATA ANALYSIS………

52

3. RESULTS AND DISCUSSION………

53

4. CONCLUSIONS………

56

Chapter 3………

58

1.LETHAL AND SUBLETHAL EFFECTS OF TWO

DIFFERENT INSECTICIDES TOWARDS A NATIVE

AND AN EXOTIC COCCINELLID……….

INTRODUCTION……….

58

1.1 COCCINELLIDS AND PESTICIDES………

60

1.1.1 COCCINELLIDS………

61

1.1.1.0 Adalia bipunctata (L.)………

61

1.1.1.1 Harmonia axyridis (Pallas)………

61

5

1.2.1 Imidacloprid………

62

1.2.2 Spinetoram………

63

2. MATERIALS AND METHODS………

65

2.1 INSECTS USED FOR THE EXPERIMENT………

65

2.1.1 A. bipunctata rearing………

65

2.1.2 H. axyridis rearing………

65

2.2 EXPERIMENT 1- ACUTE TOXICITY: mortality effects……

66

2.3 EXPERIMENT 2- CHRONIC TOXICITY: sublethal effects on

reproduction………

68

2.4 Parameters considered in the study………

69

2.5 DATA ANALYSIS………

70

3. RESULTS AND DISCUSSION………

71

3.1 EXPERIMENT 1- ACUTE TOXICITY: mortality effects………

71

3.2 EXPERIMENT 2-CHRONIC TOXICITY: sublethal effects on

reproduction………

93

6

Chapter 4………

121

1.DEVELOPMENT

OF

ENTOMOPHAGOUS

INSECTS

ON

ISOGENIC

OR

TRANSGENIC

POTATO PLANTS INFECTED BY PHYTOPHTHORA

INFESTANS

DE

BARY:

LABORATORY

EXPERIMENTS

INTRODUCTION ………

121

1.1 THE STUDY OF TRANSGENIC POTATO PLANTS EFFECTS

( AMIGA PROJECT)………

121

1.2 THE FUNGUS PHYTOPHTHORA INFESTANS DE BARY AND

THE DuRPh (GM) PLANTS………

122

1.3 EFFECTS OF GM POTATO PLANTS IN THE TRITROPHIC

INTERACTION (POTATO-APHID-PARASITOID/PREDATOR)

124

2. MATERIALS AND METHODS………

125

2.1 EXPERIMENT 1: Aphidius colemani Viereck

performance on aphids reared on Iso, Cis, Trans-genic

potato plants (Desirèe variety) infected and non-infected

by

Phytophthora

infestans

De

Bary……….

125

2.1.1 INSECTS USED FOR THE EXPERIMENT………125

7

2.1.1.2 Myzus persicae (Sulzer)……….

126

2.1.2. EXPERIMENTAL TRIAL……….

126

2.1.2.1. Description: a) Phytophthora maintenance………

129

b) Inoculation for the experiment………

129

2.1.2.2. Parameters considered in the study……….

130

2. 3 DATA ANALYSIS……….

131

3. RESULTS AND DISCUSSION………

131

2.2 EXPERIMENT 2: Larval development of Adalia

bipunctata (L.) reared on potato plants (Bintje variety)

infected and non-infected by Phytophthora infestans De

Bary……….

139

2.2.1 INSECTS USED FOR THE EXPERIMENT………

139

2.2.1.1 Adalia bipunctata (L.)………

139

2.2.1.2 Myzus persicae (Sulzer)……… ……

139

2.2.2. EXPERIMENTAL TRIAL………

139

2.2.2.1. Description………

139

2.2.2.2. Parameters considered in the study……….

141

2.2.3 DATA ANALYSIS………

141

3. RESULTS AND DISCUSSION………..

142

8

4.1 EXPERIMENT 1………

144

4.2 EXPERIMENT 2………..

144

ACKNOWLEDGEMENTS………

145

REFERENCES

CITED……….

146

9

TITLE:

NATURAL AND ANTHROPOGENIC FACTORS

AFFECTING THE LIFE CYCLE OF EXOTIC AND

NATIVE INSECT SPECIES

GENERAL INTRODUCTION

The approach of the modern agricultural method foresees the use of pesticides not as the only one system for the defense of crops, but includes the action of natural enemies of the pest agents. As in these last years there is a better sensitivity and awareness towards the importance of health of the planet, this system permits to operate in agriculture reducing the environmental impact.

My Phd thesis is the result of three years of work mostly focused on the study of strategies for a sustainable control towards exotic insect species introduced and established in Italy. In particular, the first two chapters are focused on the relationships between alien species and indigenous parasitoids, in order to evaluate the action of these entomophagous insects as potential natural enemies of the target alien pests.

In the first chapter, I compared, in laboratory conditions, the longevity and the reproduction capacity of two coccinellid species, the exotic Harmonia axyridis (Pallas) and the native Adalia

bipunctata (L.) after the exposure to the indigenous parasitoid Dinocampus coccinellae (Schrank)

(Hymenoptera Braconidae). The aim was the evaluation of the effects induced by the parasitoid on the fitness of coccinellid females, with a particular stress on the Asian H. axyridis. This exotic species was introduced some years ago in Europe and reared in biofactories for augmentative biological control purpose, but now, for its voracity and prolificacy, it is even considered by some

10

entomologists as an invasive pest so that it is no longer commercialized in Europe (Burgio et al., 2008; Kenis et al., 2008; Lombaert et al., 2014).

The second chapter is focused on the assessment of the parasitization effect of the native dipteran tachinid Exorista larvarum (L.) on the exotic box tree moth Cydalima perspectalis (Walker). This lepidopteran is one of the most recent exotic pests introduced in the European habitats (Kruger, 2008). In the experiment this alien species was compared with the factitious laboratory host

Galleria mellonella (L.). This latter species is not a natural host of E. larvarum , but in laboratory

conditions, it can support its maintenance and during the experiment acted as control. The aim was to check the possibility for Cydalima larvae to be accepted and/or successfully parasitized by the tachinid, with a complete development of the parasitoid through formation of puparia and emergence of adults.

In the third chapter I studied the lethal and sublethal effects of two different insecticides - widely used in agriculture in orchards and vegetables - on the two coccinellid species H. axyridis and A.

bipunctata. The products selected were Imidacloprid, a neonicotinoid, and Spinetoram, a compound

derived by toxins produced by the bacterium Saccharopolyspora spinosa (Mertz and Yao, 1990). The aim was to evaluate the acute and long-term effects of the two different insecticides on these insect predators with standardized methods and their fitness in laboratory (Stark and Banks, 2003). The final purpose was to investigate the laboratory response of the two coccinellid species to the two insecticides and mostly, to observe the effects of the treatments on the exotic ladybird, considered invasive. The results obtained in this laboratory research were also supposed to be a starting point in the perspective of a future study performed under field conditions.

The studies were performed in the framework of the GEISCA Project (Globalization Exotic Insects Sustainable Control Agro-forestry ecosystems) targeted at the sustainable control of exotic species, especially (but not exclusively) with respect to the action of native entomophagous insects (Maini et al., 2010).

The last part and fourth chapter of my thesis is related to my abroad experience. I carried out two laboratory experiments at the Department of Plant Sciences at Wageningen University (The Netherlands). The study was inserted in the AMIGA Project (Assessing and Monitoring The Impacts of Genetically Modified plants in Agro-Ecosystems) that is focused on the evaluation of the risk due to genetically modified organisms in the environment, following protocols set by the European Food Safety Autority (AMIGA Collaborative project proposal, 2011). In this study it was evaluated the impact of GM potato plants, resistant to the fungus Phytophthora infestans de Bary,

11

towards the tritrophic system plant-herbivorous-entomophagous insects. The aim of the first experiment was to evaluate the impact of these GM plants on the development time of the hymenopteran braconid Aphidius colemani Viereck. The parasitoid was, maintained on the green peach aphid Myzus persicae Sulzer reared on trans-genic and cis-genic potato plants infected by

Phytophthora compared with the development time tested on plants non infected by the fungus.

In the second experiment it was evaluated the development time of A. bipunctata ( from the third instar until the adult stage) fed on Myzus, that was reared on non- GM potato plants. The aim was to compare the development time occurred on plants infested and non-infested by P. infestans and to observe some effects on the third trophic level, represented by this coccinellid predator.

12

Chapter 1

1. LONGEVITY AND REPRODUCTION

CAPACITY OF TWO COCCINELLID SPECIES

PARASITIZED BY DINOCAMPUS

COCCINELLAE (SCHRANK) (HYMENOPTERA:

BRACONIDAE)

INTRODUCTION

The application of biological control often refers to the use of entomophagous insects (predators and parasitoids). These beneficial organisms may also be introduced from another Country for the biocontrol of phytophagous insects come from the same exotic Country (classical biological control) or when they play a role of high containment towards indigenous pests (Lockwood,1993) (neoclassical biological control). When an alien insect succeeds to survive and reproduce in the new habitat, it may spread and very often its populations may constitute a threat, since in the new eco-system natural enemies lack. Sometimes, also the exotic insects introduced as natural enemies against pest agents may represent this kind of problem.

In this study the exotic species that was considered as beneficial and subsequently a potential competitor towards indigenous natural enemies is the coccinellid Harmonia axyridis (Pallas). This aphid predator was tested in comparison with the native species Adalia bipunctata (L.). Both of them are Coleoptera Coccinellidae.

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Historically, ladybirds have had importance as biocontrol agents. Some of them were also introduced from other countries to solve huge pest infestations in the field (e.g. the Australian coccinellid Rodolia cardinalis Mulsant was introduced in California in 1888by C.V. Riley in citrus orchards against the cushiony scale insect Icerya purchasi Maskell; the first big action of biological control recorded (Roy and Wajnberg, 2007).

Harmonia axyridis, the multicolored Asian lady beetle is most likely native of a region that extends

from Altai Mountains to the Pacific Coast and from southern Siberia to southern China (Koch, 2003). It is also called Halloween beetle for massal groups of this coccinellid that is possible to observe in late October (Halloween time) in North America (Koch, 2003).

This polyphagous species feeds on aphids, Tetranychidae, Psillidae, Coccoidea, Curculionidae, Lepidoptera and first stages of Chrysomelidae, but also nectar and pollen (Koch, 2003). This coccinellid is so voracious that in the larval stages can consume from 100 to 400 aphids and the consumption of preys increases with the amount ofpreys and their degree of aggregation (Koch, 2003). For this reasons and for its adaptability to several environments and crop systems, H.

axyridis has been considered an optimal biocontrol agent. The case of Harmonia is the example of

how an entomophagous insect was introduced and used for pest control in a situation in which the native species could not contain enough the degree of infestation, not for the specific control of alien species in which it was requested the action of an exotic natural enemy.

The first introduction as pest control agent was in the USA in 1916 using H. axyridis of Japanese origin (Brown et al., 2011). Subsequently in 1964-65, in Europe the first releases occurred in the 1980s and the 1990s with a rapid spread, until the ascertainment of the establishment after about ten years (Roy and Wajnberg, 2007).

In Italy the “harlequin ladybird beetle” was reared in bio factories and then released in the field for the biocontrol in the 1990s, but the massal production was interrupted in 2000 for a hypothetical invasiveness towards other beneficial arthropods (Burgio et al., 2008). The coccinellid was detected for the first time in October 2006 in an urban area around Turin and again observed in the same area in 2007. Then the species was recorded in other Northern Italy regions (Burgio et al., 2008). Brown (2011) in his study, comparing the exotic species with native coccinellids, reports that in East-England H. axyridis was the most abundant species. In particular the decline of native species was likely caused by competition for prey and intraguild predation of eggs, larvae and pupae by the exotic ladybird.

14

Kovach (2004) and Ejbich (2003) explained how the case of Harmonia is emblematic. In fact, usually ladybirds are considered beneficial insects for excellence, but this exotic species is now very unpopular. This not only for the interspecific competition towards native coccinellids, but also for damages caused to humans. In fact, in autumn, it is common to find adults of this coleopteran feeding and overwintering on grapes, and then being pressed and altering the quality of wine. Furthermore, they overwinter in buildings in massive groups representing a nuisance.

About the interspecific competition, in this study it was analyzed, in laboratory conditions, the comparison in several qualitative parameters between H. axyridis and the native “two-spots ladybird” A. bipunctata that shares with the alien species the same ecological niche and in time could be substituted by Harmonia for a natural process of competitive exclusion.

The effect of a cosmopolitan parasitoid of coccinellids, Dinocampus coccinellae (Schrank) (Hymenoptera Braconidae) [first recorded in Italy on H. axyridis in 2010 (Francati, 2013)] was tested in order to ascertain if the parasitoid may represent a good natural enemy of this alien predator.

1.1 EXOTIC INSECTS AND INDIGENOUS PARASITOIDS

The introduction and establishment of exotic species in new areas are consequent of the continuous process of globalization. Furthermore, the climate change (the global warming) favours a big spread of these species until to more temperate zones. The invasion of new alien species is particularly relevant in Italy for its geographical position and for its optimal climate (Jucker and Lupi, 2011), especially in the South, also allowing survival and reproduction of subtropical species.

The Program GEISCA (Globalization Exotic Insects Sustainable Control Agro-forestry ecosystems) was developed in order to join seven research groups in Italy and an abroad collaboration for studying the phenomenon of the invasion by new hosts, developing methods for a sustainable control, mainly through the use of native natural enemies.This because the adaptation process of native natural enemies to exotic insects is only partially known. The plan was mainly focused on the role of native natural enemies in a sustainable control context towards the new intentionally or unintentionally introduced species. It has to be stressed that the success of alien species in new

15

environments is also due to the scarcity of coevolved indigenous enemies (predators and parasitoids) that can balance their fitness (enemy release hypothesis) (Keane and Crawley, 2002) (Maini et al., 2010. PRIN Project 2010-2011 prot.2010CXXHJE).

In the list of insects cited in this research project it is also inserted H. axyridis. As already said, the Asian ladybird beetle is an active aphid predator, introduced in USA and Europe in the last century for the biological control in the field and greenhouses.Then it was considered invasive because threating for the native biodiversity, in particular towards the smaller A. bipunctata, annoying for humans and being pest for grapes and wine (Kenis et al., 2008). In this framework, the aim of this

study was the assessment in laboratory of some effects induced by D. coccinellae on longevity and reproduction capacity (fitness) of Harmonia and Adalia females in order to predict the possible role of this parasitoid in a field context.

Recent studies in Italy (Dindo et al., 2014; Di Vitantonio et al., 2014) and researches in other European Countries (Berkvens et al., 2010; Kojama and Majerus, 2008) have reported a new association between Harmonia and the indigenous parasitoid in consolidation (Maini et al., 2010. PRIN Project 2010-2011 prot. 2010CXXHJE).

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1.2 Harmonia axyridis (Pallas)

Harmonia axyridis belongs to the Coleoptera order, family Coccinellidae and subfamily

Coccinellinae (Hodek et al., 2012). This coccinellid of Asian origin is common in China, South of Siberia (Koch, 2003), but also in Japan, Korea, Taiwan, Bonin and Ryukyu islands (Dobzhansky, 1933; Chapin, 1956; Iablokhoff-Khnozorian, 1982).

Initially, the description fell on the taxonomic definition Coccinella axyridis Pallas, but Jacobson and Timberlake ascribed the species under the Harmonia genus (Koch, 2003).

In Asia the “harlequin ladybird” completes only two generations per year (Sakurai et al.,1992), also in North America (LaMana and Miller,1996) and Europe (Ongagna et al., 1993). But it can arrive to have five generations per year as described by Wang (1986) and Katsoyannos et al. (1997).

The body of adults is convex shaped. The head has a facing down prognathism, well developed eyes, clubbed antennae with 11 articles and chewing mouth parts (Masutti and Zangheri, 2001). The adults measure 4.9-8.2 mm in length and 4-6.6 mm in width. The livery is variable and the head can be black, yellow or yellow with black markings. The yellowish pronotum is provided of black markings, that can be simply black spots, a black M or W-shaped, a black trapezoid (Koch, 2003). The high polymorphism in colors and pattern of elytra in adults is on genetic basis, controlled by a multi-allelic gene (Koch, 2003). Coloration and maculation can be influenced by larval diet and temperature to which pupae are exposed (Koch, 2003). The polymorphism can vary also seasonally and spatially (Koch, 2003).

Photo 1. Harmonia axyridis

17

Depending on the coloration of elytra it is possible to separate melanic forms from non-melanic forms. The melanic forms have a basic black color, and among these the typologies are:

- spectabilis Fald, with black elytra and four red spots (two per elytra); - conspicua Fald, with black elytra and two red spots (one per elytra); - aulica Fald, with black elytra and two big yellow spots.

The non-melanic forms are extremely various in number of black spots on the red, yellow, or orange elytra:

- novemdecimsignata Fald, with 19 black spots on yellowish back; - succinea Hope, with yellow elytra and without spots.

Nalepa et al. (1996) report a correspondence between the geographic distribution and the livery coloration. They underline a higher frequency of melanic forms in altitudes increasing.

Three practical systems are useful to detect the sex in the adult individuals:

- different coloration of labrum: in male the color is white or anyway lighter than the female;

- different morphology of the distal margin of the fifth abdominal sternite: in males the distal margin is concave, in females it is convex;

- chromatic difference of thoracic sternites: in male the color is lighter than female.

Furthermore, usually, females are bigger than males, but it is not an absolute condition because of the high intraspecific variety.

This is a polyphagous species, feeding on aphids, coccoides, psyllas, mites, etc. However,

Harmonia is mainly an aphidophagous coccinellid. The adult is very cold-resistant, it can survive

until -30°C (Iablokoff-Khnozorian, 1982).

In spring, with a milder temperature, at least 10°C (Ongagna et al., 1993), and the increasing of the photoperiod, the wintering individuals leave their protected site (small clefts in the soil or in the trees) and mate (LaMana and Miller, 1996).

Each female can lay 20-30 eggs per day, but in laboratory condition they can arrive to 3000 eggs with a mean of about 25 eggs per day, depending on quantity and quality of food (Hukushima and Kamei, 1970).

18

Generally yellow batches eggs are laid close to growing aphid colonies, in order to guarantee the sustainment to the progeny. This behaviour can be explained through the capacity of the females of using volatiles for an evaluation of development stage of the colony or if it is not suitable, because already parasitized by other insects (Osawa, 2000).The larvae prey from 90 to 370 aphids a day, depending on the aphid species and the instar of the larvae (Koch, 2003).

Harmonia axyridis is a holometabolous insect with four larval stages, pupa and adult (Koch, 2003).

LaMana and Miller (1998) showed that in laboratory condition, at 26°C and with a diet of

Acyrthosiphon pisum Harris the day for each phase are: egg 2.8 days, first instar larva 2.5 days,

second instar 1.5 days, third instar 1.8, the fourth instar 4.4 days, the pupa 4.5 days, 20 days totally. The eggs are yellow when freshly laid and become grey-dark before the hatching. Usually, they are laid glued to a leave, or another substrate. Larvae are very different from adults. The elongated body is covered with tubercles “scoli” and in late instars also orange bands appear. They have a chewing mouth part and start to prey immediately after the egg hatching (Koch, 2003).

At the first stages, they suck the internal fluids of preys, but with the dimension increasing they also eat solid components. Larvae of the second instar are the most voracious, but in the subsequent phases the aphids request decreases (Koch., 2003).

A frequent phenomenon in Harmonia, but also in other coccinellids, is the cannibalism. This is a survival system in condition of lack of prey. The sibling cannibalism occurs when the larvae feed on eggs not yet hatched from the same batch; the non-sibling cannibalism occurs in case larvae feed on eggs of different batches. The cannibalism is inversely related to aphid density (Burgio et al., 2002). The sibling cannibalism is more common because often the close larvae are in contact to each other. Harmonia larvae, however, prefer the non-sibling cannibalism, for the capacity to recognize relative larvae. This evolutionary strategy allow the survival of the species (Joseph et al.,1999; Michaud, 2003).

After the fourth instar, the transformation in pupa occurs. The pupa is fragile and vulnerable because the body is fixed at the substrate and exposed to external attacks. The stage of pupa is not completely immobile. In fact, if annoyed, the pupa makes shot of defense (Majerus and Kearns, 1989; Eisner and Eisner,1992). The newly emerged adult breaks the involucre and its coloration is very clear and susceptible to the external adversity because not yet sclerified.

In nature, Harmonia is a predator, but it has several natural enemies, both in the original area of distribution and also in the introduction areas. Among these, the braconid Dinocampus coccinellae

19

(Schrank), the fungi Beauveriabassiana (Bals.Criv.) and Hesperomyces virescens (Thaxter). Two kinds of defence systems may be adopted by coccinellids, including Harmonia: 1) Reflected Autohemorrhage, exudation of hemolymph of acrid smell and 2) Thanatosis, immobility that mimics death (Hodek et al., 2012).

1.3 Adalia bipunctata (L.)

Also known with the appellative of "Two spot Ladybird," this species, belonging to the subfamily Coccinellinae, is widespread in Europe and Central Asia, imported later in North America (Hodek and Honek, 2013). In our habitats it actively preys on aphids present on tree species, shrubs and herbaceous plants both cultivated and wild (Pollini , 1998). Its size is about 3.5-5.5 mm with a quite variable color. The most typical chromaticity is red-orange with two black points on the elytra. Also melanic forms with reddish-orange spots are frequent. The pronotum is black with white spots on the sides, but also cream-colored with black stain shaped central M. The body is dorsally convex and ventrally flattened, head buried in the prothorax. The antennae are filiform with the last three items slightly dilated. The legs have tarsi composed of 4 short articles, of which the second and the third bilobed is very small.

Photo 2. Adalia bipunctata, adult with aphids

(from. http://bioplanet.it/it/controllo-biologicoadalia-bipunctata)

Adalia bipunctata is a multivoltine coccinellid, it performs many generations per year. In many

areas, however, it completes only one generation per year, as wintering adult undergo diapause in

sheltered areas (Hodek and Honek, 2013; Hodek et al., 2012).

After mating, each female lays her eggs in bright yellow clusters (which show a palisade structure). The eggs are laid on the undersides of leaves (500-800 per year, depending on the prey species) (Hodek and Honek, 2013). The larval stages are 4 and they are followed by the pupal phase, which later emerges as adult. The larvae are black with white and yellow spots and are very voracious

20

preying about one hundred aphids per day, while for adults the daily ration is around 50 individuals (Hodek and Honek, 2013).

1.4 Myzus persicae (Sulzer)

Myzus persicae (Sulzer, 1776), the green peach aphid, is a tinygreen aphid (1.5-2 mm length)

belonging to the order Hemiptera and family Aphididae. Other possible primary hosts are Prunus

davidiana (Carrière) Franch. and Prunus serotina Ehrh. The origin is Palearctic, currently is

widespread worldwide (Pollini, 2013). The overwintering egg is laid on the peach tree Prunus

persica (L.) Batsch (the main primary host), where the aphid also completes some spring

generations. From June migrant forms appear and move onto herbaceous species (secondary hosts), both cultivated and wild (about 400 species belonging to different families including Cruciferae, Umbelliferae, Compositae, Solanaceae, Chenopodiaceae). In late summer, winged males and fall migrant females are produced and fly back to peach trees. On these, the fall migrant females produce wingless egg-laying females which mate with the males and lay the overwintering egg (Pollini, 2013).

Photo 3: Myzus persicae

(from http://www.nbair.res.in/Aphids/images/Myzuspersicae/Myzus%20persicae%20(10).jpg)

Gatehouse et al. (1996) reports that the importance of this pest is due to its worldwide distribution, its polyphagous nature and to the fact that it is a vector of over a hundred kinds of plant viruses. M.

persicae causes several damages on potato, sugar beet and brassicas, and in glasshouses where it

21

1.5 Dinocampus coccinellae (Schrank)

Dinocampus coccinellae (Schrank) is a wasp belonging to the Order of Hymenoptera, Family

Braconidae, Subfamily Euphorinae. The species is cosmopolitan, except for the Antarctic Continent. The origin of distribution is uncertain (Balduf, 1926), maybe Palearctic or maybe Ethiopian and accidentally introduced in new Countries with ladybirds released for the biological control. For Timberlake (1918) the wasp probably reached Hawaii islands with the host Olla

v-nigrum (Mulsant); for Gourlay (1930) it was introduced in New Zeland with the other control agent Coccinella undecimpunctata (L.).

Dinocampus coccinellae is a solitary endoparasitoid and exclusively attacks the subfamily

Coccinellinae (Balduf, 1926). But the study of Ceryngier et al. (2012) showed that occasionally the braconid can parasitize other coccinellids subfamilies in a laboratory context. The way of reproduction is parthenogenesis, thelytokous type, that is, from non-fertilized eggs only female individuals are originated. In extremely rare cases, males were obtained (Berkvens et al., 2010). The species is multivoltine over much its distribution area (Berkvens et al.,2010), for instance 2 generations in Central Europe, 3 generations in France and until 5 generations in Italy. In general about 40 species can represent its hosts, among these, H.axyridis. In Europe one of the most favourite hosts is Coccinella septempunctata (L.) (Iperti, 1964). Francati (2013) reports that D.

coccinellae does not successfully parasitize A. bipunctata, most likely because of its small size that

does not allow the development of the larva of the parasitoid (Hodek, 1973). From the field collection, the successful attack was detected for H. axyridis, C. septempunctata, Hippodamia

variegata (Goeze). Berkvenset al. (2010) recorded for the first time the new field association

between the exotic coccinellid H. axyridis and the indigenous D. coccinellae in Europe. In Italy the new association was detected for the first time in 2010 in Emilia Romagna (Francati, 2013). Mostly, D. coccinellae oviposits into adult ladybirds, but in case of their scarcity, also in larvae and pupae (Filatova, 1974; Obrycki, 1989). The indigenous wasp is also able to choose between the female and male of H. axyridis. In fact the females are parasitized much more than males (Maeta., 1969).

Coccinellids are attacked and parasitized when are mobile; if not, the braconid stimulates the coccinellid to walk with the antennae and the ovipositor (Balduf, 1926; Walker, 1961), so that the abdomen of the host is more exposed to the action of the parasitoid.

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According to Richerson and DeLoach, (1972), the action of attack can be synthesized in three moments:

- Pursuit and search of the host without extending the ovipositor;

- Positioning of the ovipositor in the ventral part of the host between the legs - Attack with introduction of the ovipositor.

Photo 4. Dinocampus coccinellae detected an individual of H. axyridis.

(from http://bugguide.net/node/view/468476

The egg of Dinocampus is elongate and, when introduced into the coccinellid body, pedunculated. Growing, it becomes oval, increasing also in lenght and stretching four times in three days. The incubation time is about five to seven days (Balduf, 1926; Sluss, 1968).

For some authors as Oglobin (1924), three are the larval stages of D. coccinellae, but for others as Balduf (1926) the stages are four. The second molt of the insect parasitoid occurs just before the exit from the host; the mature larvae leave the coccinellid body through the membrane between the fifth and sixth or sixth and seventh urite. Then it weaves the cocoon among the legs of the host and transforms into pupa.

The coccinellid, before the leakage of the larvae from the abdominal region, becomes almost immobile and this condition can extend until death, after few days. The pupa of Dinocampus in the cocoon is well protected and can exploit the advantage furnished by the structural body and the aposematic coloration of the coccinellid as defense from natural enemies.

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Photo 5. H. axyridis on pea shrub infested by aphids and with cocoon of D. coccinellae emerged from its abdomen. Entomology archive.

The wasp, when adult, leaves the cocoon chewing the apex, and each female is already prepared to attack a possible host. Each adult female emerged, in fact, is ready to oviposit.

Obrycki and Tauber (1978) detected as a temperature increasing, from 15.6°C to 26.7°C can induce a decrease of the mean time of the larval development from 47.9 to 16.3 days; a decrease of the pupal stage from 20.8 to 7.1 days and the total development time from egg to adult, from 65.8 to 23.3 days. They also observed that the development time is influenced by the host species and by the parasitized stage.

In laboratory, D. coccinellae can die for starvation in 3.6 days, if in condition of 27°C , but its longevity can increase until 20-25 days if maintained at 19°C and fed on a solution with honey or sugar (Filatova, 1974).

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2. MATERIALS AND METHODS

2.1 INSECTS USED FOR THE EXPERIMENT

The laboratory experimental trial took place at the Entomology area in the Department of Agricultural Science, University of Bologna. It started in March 2013 and ended in early July 2013. The insects used in the experiments were reared in climate rooms located in the basement of the Entomology area.

2.1.1 H. axyridis, A. bipunctata and M. persicae rearing.

Both the coccinellid species used for tests came from the rearing maintained in the climate cells at T: 25±1°C, 70±10% H.R., 16L: 8D photoperiod (Lanzoni et al., 2004); the colonies were started from individuals collected in a biological orchard and in the educational garden of the Department. Adult males and females were kept in plexiglass cages (40x30x30 cm).

Photo 6. Cage for coccinellids rearing. Entomology archive.

The cages were provided with metal net for ventilation and a small door for internal cleaning, renewal of food and collection of eggs. For the oviposition, sheets of bubble wrap sticked to the walls with scotch tape were regularly changed. Namely,two times a week or every day, if necessary, the eggs were collected cutting out fragments of bubble wrap on which they were deposited. To avoid cannibalism events and to facilitate rearing operations, all the development cycle, from egg to adult emergence, took place in plastic boxes (30x20x10 cm) with pierced lid and covered with wire mesh fine mesh for ventilation. After the emergence that requires 20 days, the adults were

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transferred in the cage for mating and oviposition. Both adults and larval stages were fed with aphids of the Myzus persicae species, reared on pea buds. For the rearing of M. persicae, twice a week from 12 to 18 plastic boxes, each containing 3 bowls filled with expanded perlite on which pea seeds were laid, were used. Pea seeds were then covered with expanded perlite and watered with 500 mL of water put at the bottom of the box.

Photo 7. Box for sowing of pea. Entomology archive.

Boxes with bowls were kept in the climate rooms at condition of T: 20±1°C, 75% H.R., 16:L 8:D photoperiod. Tuesdays and Fridays were generally the days of pea seed sowing and transfer of aphids from old infested seedlings to new shoots. These last were used as food for the two coccinellids species when necessary. Boxes and bowls were washed and sterilized with hypochlorite at the end of their use.

2.1.2 D. coccinellae rearing.

The parasitoid was reared in a different climate room (but with the same climate conditions for the coccinellids, see above), located in the opposite area of the Department in order to avoid the risk of a possible escape and contamination of the coccinellids rearing.

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The rearing of D. coccinellae started in 2010 from specimens emerged from adult individuals of H.

axyridis collected in the field and maintained following techniques described by Francati (2013).

The maintenance was achieved exposing newly emerged adults of H. axyridis to the braconid wasp (in relation 1 adult parasitoid: 10 adults of Harmonia) and keeping them in the same plexiglass cage for a time of 2 hours. The operation was weekly repeated for steadily obtaining new parasitoids ready to use in the experiments. After the exposition to the Dinocampus, the coccinellids were collected and put in plastic boxes; they were fed for about 20 days (the mean time for the parasitoid cocoon emergence) with an artificial diet containing pork liver (Sighinolfi et al., 2008), using the Francati (2013) technique. The successful parasitization occurs in case of emergence of the cocoon under the abdomen of the coccinellid. All found cocoons were taken and kept in the same cage of the adult wasps, for a continuous substitution of the old individuals. The adults were fed with honey drops taken with a small and plastic fork and put on an oil-paper strip, then glued on the internal wall of the cage.

2.2 EXPERIMENTAL TRIALS

2.2.1 Parasitoid performance test.

For testing the parasitoid performance, coccinellids obtained from the mass rearing were used. Firstly, in order to obtain adults for the exposition, pupae from the rearing were taken and individually put into small see-through plastic cylinders (5 cm height, 4 cm diameter). This operation was important for checking the emergence date, the sex of each individual, as well as the impossibility of a randomly mating before the exposition or the pair composition. Females of H.

axyridis and A. bipunctata, emerged a maximum of 4 days before, were separately exposed to D. coccinellae into microperforated and see-through plastic cylinders (20 cm height, 9 cm diameter) in

relation 1 parasitoid: 1 coccinellid female for 30 minutes, as described by Francati (2013). The individuals of D. coccinellae used for the test came from the rearing and were at maximum 72 hours old to avoid shortage of performance for old age.

Then, these coccinellid females were mated with coetaneous non-exposed males and kept into small cylinders (8 cm height, 6 cm diameter) containing strips of bubble wrap for collecting eggs. Pea shoots infested by aphids (M. persicae) were daily furnished as food to the couples.

For the experiment 80 couples were totally used; they were subdivided as follows: 40 of H. axyridis (20 couples with exposed females and 20 with non-exposed females, as control) and 40 of A.

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bipunctata (20 couples with exposed females and 20 with non-exposed females, as control). Each

couple was marked with a code. The eggs laid by the females were daily collected, counted and kept in petri-dishes on which the date of oviposition and the parent couple code were reported.

Ephestia kueniella eggs (Zeller) furnished by Biotop (Cape d’Antibes, France) were introduced to

feed the emerged larvae and to avoid to lose data for the cannibalism that they might have exerted on each other.

2.2.2 Parameters considered in the study

- The longevity of adult females from emergence (in days); - The pre-oviposition time (in days);

- The oviposition time (in days);

- The eggs laid in the first 10 days (E10)(Ferran et al., 1998 Sighinolfi et al., 2008); - The eggs laid for the total time considered (=24 days, see explanation below) (E tot); - The fertility (=% of hatched eggs) in the first 10 days;

- The total fertility.

2.3 DATA ANALYSIS

The data analysis was performed with a factorial ANOVA (2x2), in which the first factor was the coccinellid species (Harmonia/Adalia) and the second factor was the exposition of the coccinellid to the Dinocampus (exposed/non-exposed). When the data were not homogeneous, they were transformed for the analysis using the radq or the log10 transformation. When heteroscedasticity occurred despite transformation (oviposition time) the data were analyzed using the Kruskal-Wallis non parametric test, considering the factors “species” and “exposition” separately. The % values were ASN transformed for the analysis (Zar, 2010). The % parasitization (n. of formed cocoons/ n. of exposed females x100) was also considered.

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3. RESULTS AND DISCUSSION

An immediate result of the experiment carried out was that the larvae of the braconid parasitoid completed the development until the pupal cocoon formation, into 4 females on 20 H. axyridis exposed. In the exotic species it was thus attained the 20% successful parasitization.

In no female of the exposed A. bipunctata the development of D. coccinellae was completed. The exposition and the likely partial development of the parasitoid in the hosts of both the species, however, produced some effects as shown in the tables below.

Table 1a. Biological parameters referred to the coccinellids H. axyridis (H.a) and A. bipunctata

(A.b.) exposed or not-exposed to the parasitoid D. coccinellae as related to the combination of the factors “coccinellid species” and “ exposition to the parasitoid”(means±SE). The number of couples (=replicates) are given ( ) above the means. Original number of couples = 20 per thesis

Parameters Coccinellid species

Exposition to the parasitoid ANOVA Results

Yes No Effectof the host species Effect of the exposition Interaction H.a. (20) 20±0.7 (20) 22.2±0.9 F= 0.03 F=3.44 F=0.3

29 Female Longevity within 24 days from emergence (days) gl= 1,76 gl= 1,76 gl= 1,76 P= 0.87 P=0.07 P=0.58 A.b. (20) 20.7±1.2 (20) 21.9±0.8 Pre-oviposition time H.a. (18) 6.28±0.8 (19) 5.11±0.4 F= 13.78 F=1,96 0.59 gl=1,71 gl=1,71 gl=1,71 P=0.0004** 0.17 0.45 A.b. (19) 4.21±0.3 (19) 3.95±0.3 Oviposition time (days) (non parametric test of Kruskal-Wallis, for non homogeneous data, also with transformation) H.a. (18) 9.1±1.43 (19) 16.6±0.72 H=3.65 H=14.75 N=75 N=75 P=0.06 P=0.0001** A.b. (19) 12.9±1.5 (19) 17.1±0.9 Number of eggs laid in 10 gg (E10) (data transformed

for the analysis in log10) H.a. (20) 149.1±38.1 (20) 308.9±29.2 F=0.06 F=6.66 F=2.54 gl= 1,76 gl= 1,76 gl= 1,76 P=0.8 P=0.01* P=0.11 A.b. (20) 143.8±24.2 (20) 173.5±20.6 H.a. (20) 211.7±62.1 (20) 484.6±55.81 F=0.07 F=8.6 F=2.66 gl= 1,76 gl= 1,76 gl= 1,76 P=0.8 P=0.004** P=0.11

30 Number of total eggs (=laid within the 24 days) (data transformed

for the analysis in log10) A.b. (20) 230.5±42.6 (20) 289.7±31.5 Fertility referred to E10 (= n.larvae/n.laid eggs in 10 days x100) (data transformed

for the analysis in ASN) H.a. (18) 19.3±4.5 (19) 35.4±4.1 F=5.87 F=9.25 F=1.02 gl=1,71 gl=1,71 gl=1,71 P=0.02* P=0.003** P=0.32 A.b. (19) 13.7±3.3 (19) 20.6±3.7 Fertility referred to total eggs (n.larvae/n.total eggs x100) (data transformed

for the analysis in ASN) H.a. (18) 19.7±4.8 (19) 34.9±3.8 F=6.08 F=8.62 F=1.34 gl= 1,71 gl= 1,71 gl= 1,71 P=0.02* P=0.004** P=0.25 A.b. (19) 13.8±2.9 (19) 19.6±3.5

Table 1a. The parameters considered in the table refer to all the total 80 couples of the two coccinellid species in which the females were exposed or non-exposed to the parasitoid. The longevity was the elapsed time (in days) between the emergence of the female and its death. The observation was kept until the 24th day following the emergence, because within this time the successful parasitization (= cocoon emergence) could occur (Francati, 2013). In this case, as usual, the cocoon appeared after about 17 days.

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Table 1b. Biological parameters referred to the coccinellids H. axyridis (H.a) and A. bipunctata

(A.b.) exposed or non-exposed to the parasitoid D. coccinellae as related to the combination of the factors “coccinellid species” and “ exposition to the parasitoid”(means±SE). The number of couples (=replicates) are given ( ) above the means. For the exposed couples, only those which did not produce parasitoid cocoons (=unsuccessfully exposed) were originally considered.

Parameters Coccinellid species

Exposition to the parasitoid (coccinellids from which the

cocoonhasnotemerged) ANOVAResults Yes No Effect of the hostspecies Effect of theparasitizati on Interaction Longevity (days) H.a. (16) 20.1±0.9 (20) 22.2±0.9 F= 0.02 F=2.97 F=0.23 gl= 1,72 gl= 1,72 gl= 1,72 P= 0.9 P=0.09 P=0.63 A.b. (20) 20.7±1.2 (20) 21.9±0.8 Pre-oviposition time (days) H.a. (14) 5.93±0.97 (19) 5.11±0.4 F= 8.99 F=1.3 0.34 gl=1,67 gl=1,67 gl=1,67 P=0.004** 0.26 0.56 A.b. (19) 4.21±0.3 (19) 3.95±0.3

32 Oviposition time (non parametric test of Kruskal-Wallis, for non homogeneous data, also with transformatio n) H.a. (14) 10.1±1.7 (19) 16.6±0.72 H=1.91 H=13.01 N=71 N=71 P=0.17 P=0.0003** A.b. (19) 12.9±1.5 (19) 17.1±0.9 Number of laid eggs in 10 days (E10) (data transformed for the analysis in log10) H.a. (16) 171.1±45.9 (20) 308.9±29.2 F=0.07 F=5.83 F=2.2 gl= 1,72 gl= 1,72 gl= 1,72 P=0.79 P= 0.02* P=0.14 A.b. (20) 143.8±24.2 (20) 173.5±20.6 Number of total eggs (=laid for all

the life) (data transformed for the analysis in log10) H.a. (16) 249.4±74.9 (20) 484.6±55.81 F=0.03 F=7.3 F=2.18 gl= 1,72 gl= 1,72 gl= 1,72 P=0.86 P=0.009** P=0.14 A.b. (20) 230.5±42.6 (20) 289.7±31.5 Fertility referred to E10 (= H.a. (14) 22±5.4 (19) 35.4±4.1 F=7.36 F= 6.75 F=0.39 gl= 1,67 gl= 1,67 gl= 1,67 P=0.008** P=0.012* P=0.54 A.b. (19) 13.7±3.3 (19) 20.6±3.7

33 n.larvae/n.laid eggs in 10 days x100) (transformatio n in ASN) Fertility referred to total eggs (n.larvae/n. total eggs x100) (data transformed for the analysis in ASN) H.a. (14) 22.6±5.8 (19) 34.9±3.8 F=7.8 F= 6.15 F=0.56 gl= 1,67 gl= 1,67 gl= 1,67 P=0.007** P=0.02* P=0.46 A.b. (19) 13.8±2.9 (19) 19.6±3.5

Table 1b. The parameters considered in the table refer to the couples of the two coccinellid species in which the females were exposed or non-exposed to the parasitoidand in which the cocoon did not emerge. The longevity was the elapsed time (in days) between the emergence of the female and its death. The observation was kept until the 24th day following the emergence, because within this time the successful parasitization (= cocoon emergence) occurs (Francati, 2013). In this case, as usual, the cocoon appears after about 17 days.

As reported in tables 1a and 1b, the longevity indicates the lifetime of the females ladybirds. In this experiment the lifetime considered was set at 24 days; in this period the emergence of the parasitoid cocoon could be expected (Francati, 2013). In this parameter, the species, the exposure to the parasitoid and the interaction between the two factors had no significant effects.

The pre-oviposition time, that is the time between the mating and the oviposition, was influenced by the species. In fact, in Harmonia the pre-oviposition time recorded is longer then in Adalia, both for exposed and non- exposed females.

The oviposition time is the period starting from the first oviposition until the end of the observation (24 days), or the death of the female, if occurred within 24 days. In this case the exposure to the

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parasitoid had a negative effect on the parameter, reducing the days of oviposition for both the species (Kruskal-Wallis test: a) H=14.75; N=75; P=0.0001; b) H=13.01; N=71; P=0.0003). The species instead, did not influence the parameter statistically (Kruskal-Wallis test: a) H= 3.65; N=71;

P=0.0003; b) H=1.91; N=71; P=0.17).

The parameter E10 refers to the number of the eggs laid in the first 10 days from the oviposition starting. Also in this case the exposure to D. coccinellae negatively influenced the fecundity in both the species (even independently of successful parasitization). In H. axyridis the number of eggs laid by the exposed ladybirds was much lower than that of eggs laid by the controls, especially when also the successfully parasitized ladybirds were considered (table 1a and table 1b).

The Etot is the number of eggs laid for all the oviposition time considered in the study, in this case 24 days (period in which it is possible to obtain the emergence of the parasitoid cocoons). The results resemble that for E10. The exposure negatively affected the fecundity of both the species, even halving the number of eggs laid by H. axyridis (table 1a). In A. bipunctata was instead recorded a less dramatic, but significant decrease of eggs laid by the exposed females.

The fertility for E10 and Etot are percentage values obtained from the number of hatched larvae related to the laid eggs respectively in the first 10 days of oviposition and in total (until the end of observation or until the death of the female). Both the parameters showed significant effects assignable to both the species and the exposure to the parasitoid. The interaction was not significant.

It has to be stressed that similar results were obtained when the successfully parasitized (= from which the cocoon emerged) H. axyridis were either included (table 1a) or excluded (table 1b) from the analysis. The incomplete parasitoid development has thus produced some effects at least on fecundity and fertility. The number of successfully parasitized Harmonia was, however, very low and no cocoons were obtained from Adalia.

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Graph 1.The mean values of fecundity (=number of eggs laid) of H. axyridis and A. bipunctata

exposed (E) and not- exposed (C = Control) to D. coccinellae are reported in the graph below.

Graph 1. Significant effect of the factor “exposure to the parasitoid” for the parameters E10 and Etot (P=0.01 and P=0.004).

149,1 308,85 143,75 173,5 211,7 484,6 230,5 289,7 0 100 200 300 400 500 600 700 800 900 HE HC AE AC Etot E10

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The results of the fecundity in Harmonia and Adalia exposed and notexposed to Dinocampus, reported in the graph, show the impact of the exposure. In Harmonia a considerable difference between the exposed and not-exposed females occurred, more than in Adalia.

Graph 2. The mean values of fertility (=number of hatched larvae/laid eggs) of H. axyridis and

A.bipunctata exposed (E) and non- exposed (C= Control) to D. coccinellae are reported in the graph

below. 52,33 117,26 20,84 42,73 79,33 183,57 37,52 64,36 0 20 40 60 80 100 120 140 160 180 200 HE HC AE AC E10 larvae Etot larvae

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Graph 2. Significant effect of the factor “species” and the factor “exposure to the parasitoid” for both the parameters related to E10 and Etot (P=0.02 and P=0.003/0.004)

The fertility of Harmonia and Adalia was affected by both the factors and the results are visible in the graph. The values of fertility in the exposed females were always lower compared to the values in the not-exposed females. In this case also the species had a significant statistical effect.

Table 2. In the 2x2 contingency tables Harmonia and Adalia exposed or not exposed to the

parasitoid were compared in order to measure separately the effect of the exposure and the species on coccinellid longevity.. Any possible combination was tested.

Alive (at 23th day) Dead (before the 23th day) χ² (gl=1) (Yates) P n. % (±SE) (1) n. % H. axyridis Control 14 70% ± 10.5 6 30%± 10.5 6.42 0.011* H. axyridis parasitized 5 25%± 9.9 15 75%± 9.9 H. axyridis Control 14 70%± 10.5 6 30%± 10.5 0.12 0.73 A.bipunctata Control 14 70%± 10.5 6 30%± 10.5 A.bipunctata Control 14 70%± 10.5 6 30%± 10.5 A.bipunctata 12 60%± 11.2 8 40%± 11.2

38 parasitized 0.11 0.74 H. axyridis parasitized 5 25%± 9.9 15 75%± 9.9 3.68 0.055 A.bipunctata parasitized 12 60%± 11.2 8 40%± 11.2 H. axyridis Control 14 70%± 10.5 6 30%± 10.5 3.91 0.047* H. axyridis exposedbut non successfullyparasitized 5 31.3%± 11.9 11 68.7%± 11.9 A.bipunctata parasitized 12 60%± 11.2 8 40%± 11.2 1.91 0.17 H. axyridis exposedbut non successfullyparasitized 5 31.3%± 11.9 11 68.7%± 11.9 A.bipunctata Control 14 70%± 10.5 6 30%± 10.5 3.91 0.048* H. axyridis exposedbut non successfullyparasitized 5 31.3%± 11.9 11 68.7%± 11.9 H. axyridis Control 14 70% ± 10.5 6 30%± 10.5 0.11 0.74 A.bipunctata parasitized 12 60%± 11.2 8 40%± 11.2 H. axyridis parasitized 5 25%± 9.9 15 75%± 9.9 0.001 0.97 H. axyridis exposed

but non successfully parasitized

5 31.3%± 11.9 11 68.7%±

11.9

Table 2. In the 2x2 contingency tables the comparisons 2x2 between the longevity of Harmonia and Adalia exposed and not-exposed are shown. The comparison involved the alive coccinellids until the end of the observation (=24th day from the exposure) and those dead before the 24th day. The χ2 values are reported for each combine. The significant P values are marked with a star (*)

The 2x2 contingency tables showed that in all the possible combinations, significant results were recorded in case of exposed vs not exposed Harmonia. The parasitoid affected the exposed females, reducing the lifetime. The same significant result was also observed between the control and the exposed, but non successfully parasitized, females. In fact, also these females died before the 24th

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day in more cases compared to the control. The same difference is also evident in the comparison between Adalia control and Harmonia exposed but non successfully parasitized. In all the other cases the results did not show significant differences.

It is important to underline that all the Harmonia successfully parasitized (from which the cocoon emerged) died before the 24th day.

4. CONCLUSIONS

The study was performed within a research project (GEISCA) aimed at testing and demonstrating the potential of control action of Dinocampus coccinellae (Schranck) as natural enemy of the exotic coccinellid Harmonia axyridis (Pallas) compared to the native Adalia bipunctata (L.).

In the test it was evaluated which of the two coccinellid species was more successfully parasitized and if the parasitization mainly influenced longevity, pre-oviposition time, oviposition time, fecundity and fertility of the females of one or the other species.

As regards longevity, the factorial analysis of variance did not show any influence of either species or exposure. However, the 2x2 contingency tables showed that in exposed Harmonia more individuals died before the 24th day. This result suggests that the effect of D. coccinellae on this parameter deserves more research.

The exposure to the D. coccinellae did not affect the pre-oviposition time of the two species. However the exposure affected the oviposition time, reducing it in both Adalia and Harmonia. This phenomenon was probably related to the development of the parasitoid larvae in the ladybird body.

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It is also likely that the parasitoid development (if any) stopped in the exposed Adalia, because the females of this species continued to oviposit over the 10th day of the experiment. Instead, the exposed Harmonia stopped ovipositing at the 9th day. This parameter was mainly negatively affected in the exotic coccinellid.

Within the time of observation of 24 days, about the control females, Harmonia laid a bigger number of eggs than Adalia, as expected, but the oviposition time was not significant different between the species.

Some exotic ladybird were successfully parasitized, but the number of cocoons emerged from the hosts was only 4/20 with a percentage of parasitization of 20%.

The action of the parasitoid wasp negatively affected the number of laid eggs (fecundity), as well as the number of hatched larvae from the eggs (fertility) in both the coccinellid species. In this last parameter also the species influenced the hatching of the eggs. These significant differences occurred both in E10 ( first 10 days from the oviposition beginning) and in E tot (total time of oviposition until the end of observation).

The results obtained in this experiment showed that the exposure to the Dinocampus negatively affected the fitness of both the coccinellid species, but in lesser way in A. bipunctata. Probably these effects were the sum of physical and/ or physiological damage caused by the parasitoid larvae during the development into the body of the coccinellid, as observed in many host-parasitod systems (Vinson and Iwantsch, 1980; Dindo, 1987; Koyama and Majerus, 2008). This phenomenon was maybe less impactful in Adalia, which, due to its smaller size and other unknown factors, did not allow a complete development (if any) to the parasitoid. Other laboratory experimental trials demonstrated the poor fitness of native Adalia as host of D. coccinellae (Honek, 1993; Francati 2013). Due to its preference for the exotic species, the indigenous parasitoid can contribute to the containment of H. axyridis ( also with a reduction of its fitness) without representing a big threat for the small A. bipunctata.

Dinocampus coccinellae is already known as a potential natural enemy of Harmonia in different

Countries, for instance Belgium (Berkvens et al., 2010), Canada (Firlej et al., 2005) and in Italy too (Francati 2013). This study confirms its possible contribution to the control of Harmonia populations, although at little extent.

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It is important to observe that the study was carried out in laboratory conditions and in the field the situation is mitigated by many variables: the effect of Dinocampus is thus likely to be reduced. It would be desirable to extend the study in a field context.

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Chapter 2

1. PARASITIZATION EFFECT OF THE TACHINID

EXORISTA LARVARUM (L.) ON THE BOX TREE

MOTH CYDALIMA PERSPECTALIS (WALKER)

(LEPIDOPTERA: CRAMBIDAE)

INTRODUCTION

The alien species increasingly represent a great ecological and economical problem, negatively influencing the biodiversity of our habitats and also compromising our traditional landscapes (Bella, 2013). Hulme and Roy (2010) report the high threat level due to the alien insects, as one of the most dangerous groups towards the European economy. About the order Lepidoptera, more than the 70% of introductions in Europe occurred during the last century. It was also calculated that 2 new arrivals established per year between 2000 and 2007 (Lopez-Vaamonde et al., 2010). Examining 78 exotic lepidopteran species, it was shown that a bigger number came from Asia compared to the other geographic zones (Lopez-Vaamonde et al., 2010).

This progressive introduction was unintentionally increased through an increasing requirement of the commercialization of plants (Bella, 2013).

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The box tree moth Cydalima perspectalis (Walker) was one of the most recent exotic species arrived in Europe. It was recorded for the first time in 2007 in south-western Germany and the Netherlands, probably introduced with Buxus seedlings from East Asia (Kruger, 2008). In fact China, Japan, Korea and India are its original Countries (Wan et al., 2014). In this last years the species continued to spread in the rest of Europe (Nacambo, 2013). According to Nacambo et al. (2013) the insect will likely invade all the European areas, except the Northern Scotland, Northern Fenno-Scandinavia and the high mountain regions. In Italy the first record of the species was in 2010 in Lombardia region, in Como province. It probably came from Switzerland and now it is spreading across the Northern part of our Country (Bella, 2013). In Emilia Romagna the first record was in 2012, and, subsequently Tuscany and Marche regions were also colonised. The most recent Italian finding sites of Cydalima were in Catania province (Sicily), on plants of Buxus sempervirens L. “Rotundifolia” imported from Tuscany (Bella, 2013) and Perugia province (Umbria) in several gardens and nurseries during the summer 2014 (Salerno et al., 2014).

This moth is a pest of plants belonging to the genus Buxus (family Buxaceae) that counts about 90 species of primitive angiosperms, common in most tropical regions and in the Mediterranean basin (Leuthardt et al., 2013). In its original area, the insect mainly feeds on Buxus microphylla Siebold e Zucc. in Japan and in B. microphylla spp. sinica (Rehd. et Wils.) in China. It was probably introduced in the Russian Far East, since in that area Buxus spp. are not native plants (Kirpichnikova, 2005). Other plants confirmed in China and Japan as hosts of this lepidopteran are Euonymus alatus (Thunberg) Siebold, Ilex purpurea Hasskarl (Aquifoliaceae) and Euonymus

japonicus (Thunberg) (Celastraceae) (Uezumi, 1975; Shi and Hu, 2007). In the introduced habitats,

the larvae extend their diet on new varieties (Leuthardt et al., 2013). In Europe, the damages caused by C. perspectalis are particularly serious in natural areas where the native Buxus sempervirens is an essential component of a unique forest eco-system, such as the Southern part of the Massif Central in France and the Pyrenees (Di Domenico et al., 2012; Kenis et al., 2013). The box tree is a typical ornamental plant of public and private gardens (e.g. the Italian style geometric gardens), nurseries, cimiteries and parks (Bella, 2013). In Italy, the already fragmented distribution of B.

sempervirens could be seriously compromised by repeated attacks of Cydalima.

This Asian lepidopteran was inserted in EPPO Alert list in 2007, but, until 2010, none of the member Countries suggested an international action to contain this new alien. In 2011, therefore, the deletion from the Alert list followed, after a consideration of sufficient alert towards the pest (EPPO, website). Factors that can control its spread are attributable to temperature, photoperiod and humidity (Nacambo et al., 2013). In the new environments C. perspectalis seems not having natural